CDK5 genetic markers associated with galantamine response

ABSTRACT

Haplotypes in the CDK5 gene associated with cognitive response to galantamine treatment are disclosed. Compositions and methods for detecting and using these CDK5 haplotypes in a variety of clinical applications are disclosed. Such applications include articles of manufacture comprising galantamine or derivatives thereof that are approved for treating patients having one of these CDK5 haplotypes, methods and kits for predicting the response of an individual to galantamine based upon his/her haplotype profile, and methods for treating Alzheimer&#39;s patients based upon their haplotype profile.

FIELD OF THE INVENTION

This invention relates to the field of genomics and pharmacogenetics.More specifically, this invention relates to variants of the gene forcyclin-dependent kinase-5 (CDK5) and their use as predictors of anindividual's response to galantamine.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a fatal degenerative disorder of the centralnervous system that affects an estimated 3%-4% of the United Statespopulation above the age of 65 (Katzman, Arch. Neurol. 33:217-8 (1976)).AD is characterized by profound memory impairment, emotionaldisturbance, and in late stages, personality changes (Bartolucci et al.,Proteins 42:182-91 (2001)). Molecular symptoms include neuronal loss,synaptic damage, and increased levels of neurofibrillary tangles,neuritic plaques, and granulovacuolar degeneration. The reducedcognitive function seen in patients with AD are thought to be primarilyrelated to the degeneration of cholinergic neurons in the cortex andhippocampus, which results in deficits of cholinergic transmission andreduced levels of acetylcholine (Scott et al., Drugs 60(5):1095-1122(2000)). Studies have shown that AD is associated with decreased levelsof choline acetyltransferase (CHAT) and nicotinic acetylcholinereceptors (nAChRs) (Bartolucci et al., supra).

Since there is no cure for AD at the present time, current treatment forAD patients focuses on relieving some of the symptoms associated withthis disease. The major strategies revolve around increasing centralcholinergic function by elevating the transient levels of acetylcholinein the brain (cholinergic therapy). Current drugs for elevatingacetylcholine levels are AChE inhibitors, which decrease the degradationof acetylcholine in the synaptic cleft, allowing for increased neuronaltransmission, and nicotinic agonists, which directly enhance thefunction of nAChRs (Scott et al., supra; Bartolucci et al., supra).

Cholinergic therapy may also have beneficial effects for mild or minimalcognitive impairment (MCI). MCI is a condition characterized by subtlecognitive deficits not severe enough to be classified as true dementia,but in many patients represents an early stage of AD (Almkvist et al.,J. Neural Transm. Suppl. 54: 21-29 (1998)). Thus, if drug therapy toenhance cognition is started when the symptoms of dementia first appear,even before a clinical diagnosis of AD, it is possible that the onset ofAD may be delayed (Small, Hippocrates 14(9) (2000)). Other cognitivedisorders that may benefit from cholinergic therapy are vasculardementias and Lewy body dementias.

One compound that has been approved in the United States for thetreatment of mild to moderate dementia of the Alzheimer's type isgalantamine, which is a tertiary alkaloid, and marketed as Reminyl®(galantamine hydrobromide) by Janssen Pharmaceuticals (Scott et al.,Drugs 60(5):1095-1122 (2000)). Although clinical trials have establishedgalantamine's efficacy in producing significant improvement in cognitivefunction and activities of daily living in AD patients as compared toplacebo treatment (Raskind et al., Neurology 54:2261-8 (2000); Coyle etal., Biol. Psychiatry 49:289-99 (2001); Rockwood et al., J. Neurol.Nerurosurg. Psychiatry 71:589-595 (2001)), it is not clear whether ornot all patients with mild to moderate AD will ultimately demonstrate aclinically meaningful improvement in cognitive function, suggesting thatthere may be variability in an individual's response to pharmaceuticalagents to treat cognitive impairment. However, physicians currently areunable to identify patients who are at risk for reduced or lack ofefficacy of galantamine therapy, which can be expensive and is notwithout risk of side effects, with the most common side effects beingnausea, vomiting, diarrhea, dizziness and anorexia (Wilcock et al., BMJ321:1-7 (2000); Scott et al., supra). Thus it would be useful tounderstand the biological basis for the variability of response togalantamine.

Variability in the efficacy and toxicity of a number of drugs has beencorrelated with genetic variation in proteins involved in drugmetabolism (Evans et al., Science 286:487-91 (1999)). Metabolism ofgalantamine is primarily mediated by the cytochrome p-450 enzyme system,specifically the isozymes 2D6 and 3A4 (CYP2D6 and CYP3A4) (Scott et al.,supra). Poor CYP2D6 metabolizers exhibit about 25% less clearance ofgalantamine than extensive CYP2D6 metabolizers, although this differenceis not considered to be clinically relevant because the recommendeddosage regimen is to individually titrate the dose to tolerability(Reminyl® tablets prescribing information, Janssen PharmaceuticaProducts, March 2001). In addition, while several metabolites ofgalantamine inhibit AChE in vitro, their in vivo activity is notconsidered to be clinically relevant (Scott et al., supra).

Another potential source of variability of response to galantamine couldbe genetic variation in proteins involved in the etiology of AD or itsseverity, or in the mechanism of action of galantamine. For example, asmany as 70% of AD patients have a particular single nucleotidepolymorphism in the gene encoding apolipoprotein E (the ApoE4 allele)that appears to be correlated with a greater impairment of cholinergicfunction and a study with the ACHE inhibitor tacrine suggested that thepresence of this polymorphism is correlated with reduced response totreatment (Farlow et al., Neurology 50:669-77 (1998)). However, otherstudies with galantamine showed no significant difference in efficacy ofresponse between patients with zero, one or two copies of the ApoE4allele (Raskind et al., supra; Aerssens et al., poster presented at7^(th) International World Alzheimer's Congress, Jul. 9-18, 2000,Washington, D.C.).

One protein whose deregulation may be involved in neurodegenerativedisease is CDK5, a serine/threonine kinase (Zheng et al., Eur. J.Biochem. 269(18):4427-34 (2002)), which is encoded by a gene onchromosome 7q36 that consists of 12 exons. Required for normal mammaliancentral nervous system development, CDK5 is activated by itsneuron-specific activator, p35, and phosphorylates both high molecularweight neurofilaments and microtubule-associated protein tau (Hashiguchiet al., J. Biol. Chem. 277(46):4425-30 (2002)). p25, a calpain digestedtuncated form of p35, has been found to accumulate in the neurons ofAlzheimer's patients (Tseng et al., FEBS Lett. 523(1-3):58-62 (2002)).Unlike p35, p25 is not readily degraded, and the binding of p25 to CDK5constitutively activates CDK5, changes its cellular location, alters itssubstrate specificity, and increases its phosphorylation ofserine(202)/theonine(205) in tau (Hashiguchi et al., supra). Thishyperphosphorylation reduces the ability of tau to associate withmicrotubules leading to cytoskeletal disruption, morphologicdegeneration, and apoptosis (Patrick et al., Nature 402(6762):615-22(1999); Liu et al., FEBS Lett. 530(1-3):209-14 (2002)).

Recently, various haplotypes of the CHRNA2, EPHX2, and LRPAP1genes—three genes either directly or indirectly involved in the etiologyof cognitive disorders, namely AD in the case of CHRNA2 and LRPAP1, andParkinson's Disease in the case of EPHX2—were discovered to exist in acohort of Alzheimer's patients, and were discovered to be associatedwith a response to galantamine therapy. Because of its involvement inthe etiology of AD, CDK5 is a logical candidate for a determination ofwhether CDK5 haplotypes are similarly associated with response togalantamine therapy.

SUMMARY OF THE INVENTION

Accordingly, the inventors herein have discovered a set of haplotypes inthe CDK5 gene that are associated with response to galantamine. Theinventors have also discovered that the copy number of each of theseCDK5 haplotypes affects the level of galantamine response. The CDK5haplotypes are shown in Table 1 below.

TABLE 1 CDK5 Haplotypes Having Association with Response to Galantamine¹Polymorphic Site (PS) Haplotype 1 2 3 4 (1) G G (2) G G G ¹The absenceof a PS entry for a haplotype indicates that the PS is not part of themarker.

If an individual has zero copies of any of haplotypes (1)-(2) in Table1, then that individual is defined as having a “response marker I” andis more likely to respond to galantamine than an individual having onecopy of any of haplotypes (1)-(2) in Table 1, such individual beingdefined as having a “response marker II,” or two copies of any ofhaplotypes (1)-(2) in Table 1, such individual being defined as having a“response marker III.” Also, an individual having a response marker IIis more likely to respond to galantamine than an individual having aresponse marker III. Information about the composition of each ofhaplotypes (1)-(2), namely the location in the CDK5 gene of each of thepolymorphic sites (PSs), and the identity of the reference and variantallele at each PS, can be found in Table 2, shown below.

TABLE 2 Polymorphic Sites Identified in the CDK5 Gene of CaucasianIndividuals with Alzheimer's Disease Position in PS FIG. 1/SEQ ReferenceVariant Number Poly ID¹ Location ID NO: 1 Allele Allele 1 44504282promoter 1670 C G 2 44510007 intron 5 3892 G C 3 44510316 intron 9 4808A G 4 44511200 intron 9 5284 G T ¹The Poly ID is a unique identifierassigned to the indicated PS by Genaissance Pharmaceuticals, Inc., NewHaven, CT.

In addition, as described in more detail below, the inventors believethat additional haplotypes may readily be identified based on linkagedisequilibrium between any of the above CDK5 haplotypes and anotherhaplotype located in the CDK5 gene or another gene, or between an alleleat one or more of the PSs in the above haplotypes and an allele atanother PS located in the CDK5 gene or another gene. In particular, suchhaplotypes include haplotypes that are in linkage disequilibrium withany of haplotypes (1)-(2) in Table 1, hereinafter referred to as “linkedhaplotypes,” as well as “substitute haplotypes” for any of haplotypes(1)-(2) in which one or more of the polymorphic sites (PSs) in theoriginal haplotype is substituted with another PS, wherein the allele atthe substituted PS is in linkage disequilibrium with the allele at thesubstituting PS.

In one aspect, the invention provides methods and kits for determiningwhether an individual has a response marker I, a response marker II, ora response marker III. These methods and kits are useful for predictingthe expected therapeutic response of an individual to treatment withgalantamine.

In one embodiment, a method is provided for determining whether anindividual has a response marker I, a response marker II, or a responsemarker III comprising determining whether the individual has zero, one,or two copies of any of (a) haplotypes (1)-(2) in Table 1, (b) a linkedhaplotype for any of haplotypes (1)-(2) in Table 1, and (c) a substitutehaplotype for any of haplotypes (1)-(2) in Table 1.

In another embodiment of the invention, a method is provided forassigning an individual to a first, second, or third response markergroup comprising determining whether the individual has zero, one, ortwo copies of any of (a) haplotypes (1)-(2) in Table 1, (b) a linkedhaplotype for any of haplotypes (1)-(2) in Table 1, and (c) a substitutehaplotype for any of haplotypes (1)-(2) in Table 1, and assigning theindividual to a response marker group based on the copy number of thathaplotype. The individual is assigned to the first response marker groupif the individual has zero copies of any of (a) haplotypes (1)-(2) inTable 1, (b) a linked haplotype for any of haplotypes (1)-(2) in Table1, and (c) a substitute haplotype for any of haplotypes (1)-(2) in Table1, to the second response marker group if the individual has one copy ofany of (a) haplotypes (1)-(2) in Table 1, (b) a linked haplotype for anyof haplotypes (1)-(2) in Table 1, and (c) a substitute haplotype for anyof haplotypes (1)-(2) in Table 1, and to the third response marker groupif the individual has two copies of any of (a) haplotypes (1)-(2) inTable 1, (b) a linked haplotype for any of haplotypes (1)-(2) in Table1, and (c) a substitute haplotype for any of haplotypes (1)-(2) in Table1.

One embodiment of a kit for determining whether an individual has aresponse marker I, a response marker II, or a response marker IIIcomprises a set of oligonucleotides designed for identifying at leastone of the alleles present at each PS in a set of one or more PSs. Theset of one or more PSs comprises the set of one or more PSs for any ofthe haplotypes in Table 1, the set of one or more PSs for a linkedhaplotype, or the set of one or more PSs for a substitute haplotype. Ina further embodiment, the kit comprises a manual with instructions forperforming one or more reactions on a human nucleic acid sample toidentify the allele(s) present in the individual at each PS in the setand determining if the individual has a response marker I, a responsemarker II, or a response marker III based on the identified allele(s).

In yet another embodiment, the invention provides a method forpredicting an individual's response to treatment with galantamine. Themethod comprises determining whether the individual has a responsemarker I, a response marker II, or a response marker III, and making aresponse prediction based on the results of the determining step. If theindividual is determined to have a response marker I, then the responseprediction is that the individual is more likely to respond togalantamine treatment than an individual having a response marker II ora response marker III. If the individual is determined to have aresponse marker II, then the response prediction is that the individualis less likely to respond to galantamine treatment than an individualhaving a response marker I, and more likely to respond to galantaminetreatment than an individual having a response marker III. If theindividual is determined to have a response marker III, then theresponse prediction is that the individual is less likely to respond togalantamine treatment than an individual having a response marker I or aresponse marker II.

In other aspects, the invention provides (i) a method for seekingregulatory approval for marketing a galantamine pharmaceuticalformulation to a population having a cognitive disorder, wherein thepopulation is partially or wholly defined by having a response marker Ior a response marker II, (ii) an article of manufacture comprising thepharmaceutical formulation, (iii) a method for manufacturing a drugproduct comprising the pharmaceutical formulation, and (iv) a method formarketing the drug product. In a preferred embodiment, the cognitivedisorder is mild to moderate dementia of the Alzheimer's type, dementiaassociated with Parkinson's Disease, MCI, a vascular dementia or a Lewybody dementia.

The method for seeking regulatory approval comprises conducting at leastone clinical trial which comprises administering the pharmaceuticalformulation and a placebo to each of a first, second, third, and fourthtreatment group of individuals having a cognitive disorder, wherein eachindividual in the first treatment group has a response marker I, eachindividual in the second treatment group does not have a response markerI, each individual in the third treatment group has a response markerII, and each individual in the fourth treatment group has a responsemarker III, demonstrating that the first treatment group is more likelyto respond to the pharmaceutical formulation than the second treatmentgroup, demonstrating that the third treatment group is more likely torespond to the pharmaceutical formulation than the fourth treatmentgroup, and filing with a regulatory agency an application for marketingapproval of the pharmaceutical formulation with a label stating that thepharmaceutical formulation is indicated for a population having acognitive disorder, and further stating that individuals having aresponse marker I are more likely to respond to the pharmaceuticalformulation than individuals having a response marker II or a responsemarker III, and that individuals having a response marker II are morelikely to respond to the pharmaceutical formulation than individualshaving a response marker III. In preferred embodiments, the regulatoryagency is the United States Food and Drug Administration (FDA) or theEuropean Agency for the Evaluation of Medicinal Products (EMEA), or afuture equivalent of these agencies.

In one embodiment, the article of manufacture comprises thepharmaceutical formulation and at least one indicium identifying apopulation for whom the pharmaceutical formulation is indicated, whereinthe identified population is one having a cognitive disorder, andwherein the identified population is partially or wholly defined byhaving a response marker I or a response marker II, wherein a trialpopulation of individuals having a response marker I is more likely torespond to the formulation than a trial population lacking a responsemarker I, and a trial population of individuals having a response markerII is more likely to respond to the formulation than a trial populationof individuals lacking both a response marker I and a response markerII. Another embodiment of the article of manufacture comprises packagingmaterial and the pharmaceutical formulation contained within thepackaging material, wherein the packaging material comprises a labelapproved by a regulatory agency for the pharmaceutical formulation,wherein the label states that the pharmaceutical formulation isindicated for improving cognitive function in a population having acognitive disorder, wherein the population is partially or whollydefined by having a response marker I or a response marker II, andfurther stating that those members of the population having a responsemarker I are more likely to respond to the pharmaceutical formulationthan those members lacking a response marker I, and those members of thepopulation having a response marker II are more likely to respond to thepharmaceutical formulation than those members lacking both a responsemarker I and a response marker II. Preferably, the pharmaceuticalformulation comprises a galantamine compound as at least one activeingredient. The galantamine compound is selected from galantamine, agalantamine derivative, and pharmaceutically acceptable salts ofgalantamine or the galantamine derivative.

The method for manufacturing the drug product comprises combining in apackage a pharmaceutical formulation comprising a galantamine compoundas at least one active ingredient and a label which states that the drugproduct is indicated for a population having a cognitive disorder,wherein the population is partially or wholly defined by having aresponse marker I or a response marker II, wherein those members of thepopulation having a response marker I are more likely to respond to thedrug product than those members of the population lacking a responsemarker I, and those members of the population having a response markerII are more likely to respond to the drug product than those members ofthe population lacking both a response marker I and a response markerII. The galantamine compound is selected from galantamine, a galantaminederivative, and pharmaceutically acceptable salts of galantamine or thegalantamine derivative.

The method for marketing the drug product comprises promoting to atarget audience the use of the drug product for treating individuals whobelong to the defined population.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C illustrates a reference sequence for the CDK5 gene (contiguouslines; SEQ ID NO:1), with the start and stop positions of each region ofcoding sequence indicated with a bracket ([or]) and the numericalposition below the sequence and the polymorphic site(s) andpolymorphism(s) identified by Applicants in the patient cohort indicatedby the variant nucleotide positioned below the polymorphic site in thesequence.

DEFINITIONS

In the context of this disclosure, the terms below shall be defined asfollows unless otherwise indicated:

Allele—A particular form of a genetic locus, distinguished from otherforms by its particular nucleotide sequence, or one of the alternativepolymorphisms found at a polymorphic site.

Gene—A segment of DNA that contains the coding sequence for a protein,wherein the segment may include promoters, exons, introns, and otheruntranslated regions that control expression.

Genotype—An unphased 5′ to 3′ sequence of nucleotide pair(s) found at aset of one or more polymorphic sites in a locus on a pair of homologouschromosomes in an individual. As used herein, genotype includes afull-genotype and/or a sub-genotype as described below.

Genotyping—A process for determining a genotype of an individual.

Haplotype—A 5′ to 3′ sequence of nucleotides found at a set of one ormore polymorphic sites in a locus on a single chromosome from a singleindividual.

Haplotype pair—The two haplotypes found for a locus in a singleindividual.

Haplotyping—A process for determining one or more haplotypes in anindividual and includes use of family pedigrees, molecular techniquesand/or statistical inference.

Haplotype data—Information concerning one or more of the following for aspecific gene: a listing of the haplotype pairs in an individual or ineach individual in a population; a listing of the different haplotypesin a population; frequency of each haplotype in that or otherpopulations, and any known associations between one or more haplotypesand a trait.

Isolated—As applied to a biological molecule such as RNA, DNA,oligonucleotide, or protein, isolated means the molecule issubstantially free of other biological molecules such as nucleic acids,proteins, lipids, carbohydrates, or other material such as cellulardebris and growth media. Generally, the term “isolated” is not intendedto refer to a complete absence of such material or to absence of water,buffers, or salts, unless they are present in amounts that substantiallyinterfere with the methods of the present invention.

Locus—A location on a chromosome or DNA molecule corresponding to a geneor a physical or phenotypic feature, where physical features includepolymorphic sites.

Nucleotide pair—The nucleotides found at a polymorphic site on the twocopies of a chromosome from an individual.

Phased—As applied to a sequence of nucleotide pairs for two or morepolymorphic sites in a locus, phased means the combination ofnucleotides present at those polymorphic sites on a single copy of thelocus is known.

Polymorphic site (PS)—A position on a chromosome or DNA molecule atwhich at least two alternative sequences are found in a population.

Polymorphism—The sequence variation observed in an individual at apolymorphic site. Polymorphisms include nucleotide substitutions,insertions, deletions and microsatellites and may, but need not, resultin detectable differences in gene expression or protein function.

Polynucleotide—A nucleic acid molecule comprised of single-stranded RNAor DNA or comprised of complementary, double-stranded DNA.

Population Group—A group of individuals sharing a common ethnogeographicorigin.

Reference Population—A group of subjects or individuals who arepredicted to be representative of the genetic variation found in thegeneral population. Typically, the reference population represents thegenetic variation in the population at a certainty level of at least85%, preferably at least 90%, more preferably at least 95% and even morepreferably at least 99%.

Single Nucleotide Polymorphism (SNP)—Typically, the specific pair ofnucleotides observed at a single polymorphic site. In rare cases, threeor four nucleotides may be found.

Subject—A human individual whose genotypes or haplotypes or response totreatment or disease state are to be determined.

Treatment—A stimulus administered internally or externally to a subject.

Unphased—As applied to a sequence of nucleotide pairs for two or morepolymorphic sites in a locus, unphased means the combination ofnucleotides present at those polymorphic sites on a single copy of thelocus is not known.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Each response marker of the invention is a combination of a particularhaplotype and the copy number for that haplotype. Preferably, thehaplotype is one of the haplotypes shown in Table 1. The PS or PSs inthese haplotypes are referred to herein as PS1, PS2, PS3, and PS4, andare located in the CDK5 gene at positions corresponding to thoseidentified in FIG. 1/SEQ ID NO:1 (see Table 2 for summary of PS1, PS2,PS3, and PS4, and locations). In describing the PSs in the responsemarkers of the invention, reference is made to the sense strand of agene for convenience. However, as recognized by the skilled artisan,nucleic acid molecules containing a particular gene may be complementarydouble stranded molecules and thus reference to a particular site orhaplotype on the sense strand refers as well to the corresponding siteor haplotype on the complementary antisense strand. Further, referencemay be made to detecting a genetic marker or haplotype for one strandand it will be understood by the skilled artisan that this includesdetection of the complementary haplotype on the other strand.

As described in more detail in the examples below, the response markersof the invention are based on the discovery by the inventors ofassociations between certain haplotypes in the CDK5 gene and response togalantamine treatment in a cohort of individuals diagnosed withAlzheimer's Disease.

In particular, the inventors herein discovered that a haplotypecomprising guanine at PS3 and guanine at PS4 (haplotype (1) in Table 1)affected the response to galantamine of the patients participating inthe study. The group of patients having zero copies of this haplotypeexperienced a better response to galantamine than the patient grouphaving one copy, which experienced a better response to galantamine thanthe patient group having two copies. As used herein, the terms“galantamine response” and “response to galantamine,” are intended torefer to the change in an individual's cognitive function, preferably asmeasured by his/her score on the cognitive subscale of the Alzheimer'sDisease Assessment (ADAS-cog) (Rosen et al., Am. J. Psychiatry141:1356-64 (1984); Rockwood et al., J. Neurol. Neurosurg. Psychiatry71:589-95 (2001); Tariot et al., Neurology 54:2269-76 (2000); Wilcock etal., BMJ 321:1-7 (2000)) following galantamine treatment/administration.The ADAS-cog measures cognitive function, including spoken languageability, comprehension of spoken language, recall of test instructions,word-finding difficulty in spontaneous speech, following commands,naming objects and fingers, constructional praxis, ideational praxis,orientation, word-recall task and word-recognition task (Alzheimer'sInsights Online, Vol. 3, No. 1, 1997). With regard to the ADAS-cog, thelower the score, the better the cognitive function. Thus, a downwardchange in the ADAS-cog following galantamine treatment/administrationindicates a “good” or “positive” or “better” response to galantamine(or, simply, “response”), and an upward change, or no change, in theADAS-cog following galantamine treatment/administration indicates a“bad” or “negative” or “worse” response to galantamine (or, simply,“non-reponse”). Additionally, an individual's response to galantaminemay be measured by other scientifically accepted rating scales forcognitive function, including, but not limited to, Behavioral Pathologyin Alzheimer's Disease Rating Scale (BEHAVE-AD), Blessed Test, CANTAB(CAmbridge Neuropsychological Test Automated Battery), CERAD (TheConsortium to Establish a Registry for Alzheimer's Disease) Clinical andNeuropsychological Tests, Clock Draw Test, Cornell Scale for Depressionin Dementia (CSDD), Geriatric Depression Scale (GDS), Mini Mental StateExam (MMSE), Neuropsychiatric Inventory (NPI), and The 7 Minute Screen.

Moreover, as shown in Tables 10A, 10B, and 10C below, the differenteffect of copy number of haplotype (1) on galantamine response isstatistically significant. Therefore, this haplotype, in combinationwith the haplotype copy number, can be used to differentiate thegalantamine response that might be observed in an individual or a trialpopulation after treatment with galantamine. Consequently, zero copiesof haplotype (1) in Table 1 is referred to herein as a response markerI, one copy of haplotype (1) in Table 1 is referred to herein as aresponse marker II, and two copies of haplotype (1) in Table 1 isreferred to herein as a response marker III.

In addition, the skilled artisan would expect that there might beadditional PSs in the CDK5 gene or elsewhere on chromosome 7, wherein anallele at that PS is in high linkage disequilibrium (LD) with an alleleat one or more of the PSs in the haplotypes comprising a response markerI, a response marker II, or a response marker III. Two particularalleles at different PSs are said to be in LD if the presence of theallele at one of the sites tends to predict the presence of the alleleat the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17(1999)). One of the most frequently used measures of linkagedisequilibrium is Δ², which is calculated using the formula described byDevlin et al. (Genomics 29(2):311-22 (1995)). Δ² is the measure of howwell an allele X at a first PS predicts the occurrence of an allele Y ata second PS on the same chromosome. The measure only reaches 1.0 whenthe prediction is perfect (e.g., X if and only if Y).

Thus, the skilled artisan would expect that all of the embodiments ofthe invention described herein may frequently be practiced bysubstituting any (or all) of the specifically identified CDK5 PSs in aresponse marker with another PS, wherein an allele at the substituted PSis in LD with an allele at the “substituting” PS. This “substituting” PSmay be one that is currently known or subsequently discovered and may bepresent in the CDK5 gene, in a genomic region of about 100 kilobasesspanning the CDK5 gene, or elsewhere on chromosome 7.

Further, the inventors contemplate that there will be other haplotypesin the CDK5 gene or elsewhere on chromosome 7 that are in LD with one ormore of the haplotypes in Table 1 that would therefore also bepredictive of galantamine response. Preferably, the linked haplotype ispresent in the CDK5 gene or in a genomic region of about 100 kilobasesspanning the CDK5 gene. The linkage disequilibrium between thehaplotypes in Table 1 and such linked haplotypes can also be measuredusing Δ².

In preferred embodiments, the linkage disequilibrium between an alleleat a polymorphic site in any of the haplotypes in Table 1 and an alleleat a “substituting” polymorphic site, or between any of the haplotypesin Table 1 and a linked haplotype, has a Δ² value, as measured in asuitable reference population, of at least 0.75, more preferably atleast 0.80, even more preferably at least 0.85 or at least 0.90, yetmore preferably at least 0.95, and most preferably 1.0. A suitablereference population for this Δ² measurement is preferably a populationfor which the distribution of its members reflects that of thepopulation of patients to be treated with galantamine. The referencepopulation may be the general population, a population usinggalantamine, a population with AD or AD risk factors, or the like.

LD patterns in genomic regions are readily determined empirically inappropriately chosen samples using various techniques known in the artfor determining whether any two alleles (either those occurring at twodifferent PSs or two haplotypes for two different multi-site loci) arein linkage disequilibrium (GENETIC DATA ANALYSIS II, Weir, SinauerAssociates, Inc. Publishers, Sunderland, Mass., 1996). The skilledartisan may readily select which method of determining LD will be bestsuited for a particular sample size and genomic region.

As described above and in the examples below, the response markers ofthe invention are associated with changes in the cognitive subscale ofthe Alzheimer's Disease Assessment Scale (ADAS-cog) in response togalantamine treatment. Thus, the invention provides a method and kit fordetermining whether an individual has a response marker I, a responsemarker II, or a response marker III. A response marker I is zero copiesof any of (a) haplotypes (1)-(2) in Table 1, (b) a linked haplotype forany of haplotypes (1)-(2) in Table 1, and (c) a substitute haplotype forany of haplotypes (1)-(2) in Table 1. A response marker II is one copyof any of (a) haplotypes (1)-(2) in Table 1, (b) a linked haplotype forany of haplotypes (1)-(2) in Table 1, and (c) a substitute haplotype forany of haplotypes (1)-(2) in Table 1. A response marker III is twocopies of any of (a) haplotypes (1)-(2) in Table 1, (b) a linkedhaplotype for any of haplotypes (1)-(2) in Table 1, and (c) a substitutehaplotype for any of haplotypes (1)-(2) in Table 1.

In one embodiment, the invention provides a method for determiningwhether an individual has a response marker I, a response marker II, ora response marker III. The method comprises determining whether theindividual has zero copies, one copy, or two copies of any of (a)haplotypes (1)-(2) in Table 1, (b) a linked haplotype for any ofhaplotypes (1)-(2) in Table 1, and (c) a substitute haplotype for any ofhaplotypes (1)-(2) in Table 1.

In some embodiments, the individual is Caucasian and may be diagnosedwith a cognitive disorder, such as mild to moderate dementia of theAlzheimer's type, dementia associated with Parkinson's Disease, MCI, avascular dementia, and Lewy body dementia, may have risk factorsassociated with a cognitive disorder, or may be a candidate fortreatment with galantamine for an alternative reason.

In another embodiment, the invention provides a method for assigning anindividual to a first, second, or third response marker group. Themethod comprises determining whether the individual has zero copies, onecopy, or two copies of any of (a) haplotypes (1)-(2) in Table 1, (b) alinked haplotype for any of haplotypes (1)-(2) in Table 1, and (c) asubstitute haplotype for any of haplotypes (1)-(2) in Table 1, andassigning the individual to the first response marker group if theindividual has zero copies of any of (a) haplotypes (1)-(2) in Table 1,(b) a linked haplotype for any of haplotypes (1)-(2) in Table 1,assigning the individual to the second response marker group if theindividual has one copy of any of (a) haplotypes (1)-(2) in Table 1, (b)a linked haplotype for any of haplotypes (1)-(2) in Table 1, and (c) asubstitute haplotype for any of haplotypes (1)-(2) in Table 1, andassigning the individual to the third response marker group if theindividual has two copies of any of (a) haplotypes (1)-(2) in Table 1,(b) a linked haplotype for any of haplotypes (1)-(2) in Table 1, and (c)a substitute haplotype for any of haplotypes (1)-(2) in Table 1.

In some embodiments, the individual is Caucasian and may be diagnosedwith a cognitive disorder, such as mild to moderate dementia of theAlzheimer's type, dementia associated with Parkinson's Disease, MCI, avascular dementia, and Lewy body dementia, may have risk factorsassociated with a cognitive disorder, or may be a candidate fortreatment with galantamine for an alternative reason.

The presence in an individual of a response marker I, a response markerII, or a response marker III may be determined by a variety of indirector direct methods well known in the art for determining haplotypes orhaplotype pairs for a set of one or more PSs in one or both copies ofthe individual's genome, including those discussed below. The genotypefor a PS in an individual may be determined by methods known in the artor as described below.

One indirect method for determining whether zero copies, one copy, ortwo copies of a haplotype is present in an individual is by predictionbased on the individual's genotype determined at one or more of the PSscomprising the haplotype and using the determined genotype at each siteto determine the haplotypes present in the individual. The presence ofzero copies, one copy, or two copies of a haplotype of interest can bedetermined by visual inspection of the alleles at the PS that comprisethe haplotype. The haplotype pair is assigned by comparing theindividual's genotype with the genotypes at the same set of PScorresponding to the haplotype pairs known to exist in the generalpopulation or in a specific population group or to the haplotype pairsthat are theoretically possible based on the alternative allelespossible at each PS, and determining which haplotype pair is most likelyto exist in the individual.

In a related indirect haplotyping method, the presence in an individualof zero copies, one copy, or two copies of a haplotype is predicted fromthe individual's genotype for a set of PSs comprising the selectedhaplotype using information on haplotype pairs known to exist in areference population. In one embodiment, this haplotype pair predictionmethod comprises identifying a genotype for the individual at the set ofPSs comprising the selected haplotype, accessing data containinghaplotype pairs identified in a reference population for a set of PSscomprising the PSs of the selected haplotype, and assigning to theindividual a haplotype pair that is consistent with the individual'sgenotype. Whether the individual has a response marker I, a responsemarker II, or a response marker III can be subsequently determined basedon the assigned haplotype pair. The haplotype pair can be assigned bycomparing the individual's genotype with the genotypes corresponding tothe haplotype pairs known to exist in the general population or in aspecific population group, and determining which haplotype pair isconsistent with the genotype of the individual. In some embodiments, thecomparing step may be performed by visual inspection. When the genotypeof the individual is consistent with more than one haplotype pair,frequency data may be used to determine which of these haplotype pairsis most likely to be present in the individual. If a particularhaplotype pair consistent with the genotype of the individual is morefrequent in the reference population than other pairs consistent withthe genotype, then that haplotype pair with the highest frequency is themost likely to be present in the individual. The haplotype pairfrequency data used in this determination is preferably for a referencepopulation comprising the same ethnogeographic group as the individual.This determination may also be performed in some embodiments by visualinspection. In other embodiments, the comparison may be made by acomputer-implemented algorithm with the genotype of the individual andthe reference haplotype data stored in computer-readable formats. Forexample, as described in WO 01/80156, one computer-implemented algorithmto perform this comparison entails enumerating all possible haplotypepairs which are consistent with the genotype, accessing data containinghaplotype pairs frequency data determined in a reference population todetermine a probability that the individual has a possible haplotypepair, and analyzing the determined probabilities to assign a haplotypepair to the individual.

Typically, the reference population is composed of randomly selectedindividuals representing the major ethnogeographic groups of the world.A preferred reference population for use in the methods of the presentinvention consists of Caucasian individuals, the number of which ischosen based on how rare a haplotype is that one wants to be guaranteedto see. For example, if one wants to have a q % chance of not missing ahaplotype that exists in the population at a p % frequency of occurringin the reference population, the number of individuals (n) who must besampled is given by 2n=log(1−q)/log(1−p) where p and q are expressed asfractions. A preferred reference population allows the detection of anyhaplotype whose frequency is at least 10% with about 99% certainty. Aparticularly preferred reference population includes a 3-generationCaucasian family to serve as a control for checking quality ofhaplotyping procedures.

If the reference population comprises more than one ethnogeographicgroup, the frequency data for each group is examined to determinewhether it is consistent with Hardy-Weinberg equilibrium. Hardy-Weinbergequilibrium (PRINCIPLES OF POPULATION GENOMICS, 3^(rd) ed., Hartl,Sinauer Associates, Sunderland, Mass., 1997) postulates that thefrequency of finding the haplotype pair H₁/H₂ is equal top_(H-W)(H₁/H₂)=2p(H₁)p(H₂) if H₁≠H₂ and p_(H-W)(H₁/H₂)=p(H₁)p(H₂) ifH₁=H₂. A statistically significant difference between the observed andexpected haplotype frequencies could be due to one or more factorsincluding significant inbreeding in the population group, strongselective pressure on the gene, sampling bias, and/or errors in thegenotyping process. If large deviations from Hardy-Weinberg equilibriumare observed in an ethnogeographic group, the number of individuals inthat group can be increased to see if the deviation is due to a samplingbias. If a larger sample size does not reduce the difference betweenobserved and expected haplotype pair frequencies, then one may wish toconsider haplotyping the individual using a direct haplotyping methodsuch as, for example, CLASPER System™ technology ((U.S. Pat. No.5,866,404), single molecule dilution, or allele-specific long-range PCR(Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-3 (1996)).

In one embodiment of this method for predicting a haplotype pair for anindividual, the assigning step involves performing the followinganalysis. First, each of the possible haplotype pairs is compared to thehaplotype pairs in the reference population. Generally, only one of thehaplotype pairs in the reference population matches a possible haplotypepair and that pair is assigned to the individual. Occasionally, only onehaplotype represented in the reference haplotype pairs is consistentwith a possible haplotype pair for an individual, and in such cases theindividual is assigned a haplotype pair containing this known haplotypeand a new haplotype derived by subtracting the known haplotype from thepossible haplotype pair. Alternatively, the haplotype pair in anindividual may be predicted from the individual's genotype for that geneusing reported methods (e.g., Clark et al., Mol. Biol. Evol. 7:111-22(1990) or WO 01/80156) or through a commercial haplotyping service suchas offered by Genaissance Pharmaceuticals, Inc. (New Haven, Conn.). Inrare cases, either no haplotypes in the reference population areconsistent with the possible haplotype pairs, or alternatively, multiplereference haplotype pairs are consistent with the possible haplotypepairs. In such cases, the individual is preferably haplotyped using adirect molecular haplotyping method such as, for example, CLASPERSystem™ technology (U.S. Pat. No. 5,866,404), SMD, or allele-specificlong-range PCR (Michalotos-Beloin et al., supra).

Determination of the number of haplotypes present in the individual fromthe genotypes is illustrated here for haplotype (1) in Table 1. Table 3below shows the 9 (3^(n), where each of n bi-allelic polymorphic sitesmay have one of 3 different genotypes present) genotypes that may bedetected at PS3 and PS4, using both chromosomal copies from anindividual. 8 of the 9 possible genotypes for the two sites allowunambiguous determination of the number of copies of the haplotype (1)in Table 1 present in the individual. However, an individual with theG/A G/T genotype could possess one of the following genotype pairs:GG/AT, GT/AG, AG/GT, and AT/GG, and thus could have either one copy ofhaplotype (1) in Table 1 (GG/AT, AT/GG), or zero copies (GT/AG, AG/GT)of haplotype (1) in Table 1. For instances where there is ambiguity inthe haplotype pair underlying the determined genotype (i.e., when two ormore PSs are included in the haplotype), frequency information may beused to determine the most probable haplotype pair and therefore themost likely number of copies of the haplotype in the individual. If aparticular haplotype pair consistent with the genotype of the individualis more frequent in the reference population than other pairs consistentwith the genotype, then that haplotype pair with the highest frequencyis the most likely to be present in the individual. The copy number ofthe haplotype of interest in this haplotype pair can then be determinedby visual inspection of the alleles at the PS that comprise the responsemarker for each haplotype in the pair.

Alternatively, for the ambiguous genotypes, genotyping of one or moreadditional sites in CDK5 may be performed to eliminate the ambiguity indeconvoluting the haplotype pairs underlying the genotype at theparticular PSs. The skilled artisan would recognize that alleles atthese one or more additional sites would need to have sufficient linkagewith the alleles in at least one of the possible haplotypes in the pairto permit unambiguous assignment of the haplotype pair. Although thisillustration has been directed to the particular instance of determiningthe number of copies of haplotype (1) in Table 1 present in anindividual, the process would be analogous for the other haplotypesshown in Table 1, or for the linked haplotypes or substitute haplotypesfor any of the haplotypes in Table 1.

TABLE 3 Possible Copy Numbers of Haplotype (1) in Table 1 Based onGenotypes at PS3 and PS4 Copy Number of Haploytpe PS3 PS4 (1) in Table 1G/G G/G 2 G/G G/T 1 G/G T/T 0 G/A G/G 1 G/A G/T 1 or 0 G/A T/T 0 A/A G/G0 A/A G/T 0 A/A T/T 0

The individual's genotype for the desired set of PS may be determinedusing a variety of methods well-known in the art. Such methods typicallyinclude isolating from the individual a genomic DNA sample comprisingboth copies of the gene or locus of interest, amplifying from the sampleone or more target regions containing the polymorphic sites to begenotyped, and detecting the nucleotide pair present at each PS ofinterest in the amplified target region(s). It is not necessary to usethe same procedure to determine the genotype for each PS of interest.

In addition, the identity of the allele(s) present at any of the novelPSs described herein may be indirectly determined by haplotyping orgenotyping another PS having an allele that is in linkage disequilibriumwith an allele of the PS that is of interest. PSs having an allele inlinkage disequilibrium with an allele of the presently disclosed PSs maybe located in regions of the gene or in other genomic regions notexamined herein. Detection of the allele(s) present at a PS, wherein theallele is in linkage disequilibrium with an allele of the novel PSsdescribed herein may be performed by, but is not limited to, any of theabove-mentioned methods for detecting the identity of the allele at aPS.

Alternatively, the presence in an individual of a haplotype or haplotypepair for a set of PSs comprising a response marker may be determined bydirectly haplotyping at least one of the copies of the individual'sgenomic region of interest, or suitable fragment thereof, using methodsknown in the art. Such direct haplotyping methods typically involvetreating a genomic nucleic acid sample isolated from the individual in amanner that produces a hemizygous DNA sample that only has one of thetwo “copies” of the individual's genomic region which, as readilyunderstood by the skilled artisan, may be the same allele or differentalleles, amplifying from the sample one or more target regionscontaining the PSs to be genotyped, and detecting the nucleotide presentat each PS of interest in the amplified target region(s). The nucleicacid sample may be obtained using a variety of methods known in the artfor preparing hemizygous DNA samples, which include: targeted in vivocloning (TIVC) in yeast as described in WO 98/01573, U.S. Pat. No.5,866,404, and U.S. Pat. No. 5,972,614; generating hemizygous DNAtargets using an allele specific oligonucleotide in combination withprimer extension and exonuclease degradation as described in U.S. Pat.No. 5,972,614; single molecule dilution (SMD) as described in Ruaño etal., Proc. Natl. Acad. Sci. 87:6296-300 (1990); and allele specific PCR(Ruaño et al., Nucl. Acids Res. 17:8392 (1989); Ruaño et al., Nucl.Acids Res. 19:6877-82 (1991); Michalatos-Beloin et al., supra).

As will be readily appreciated by those skilled in the art, anyindividual clone will typically only provide haplotype information onone of the two genomic copies present in an individual. If haplotypeinformation is desired for the individual's other copy, additionalclones will usually need to be examined. Typically, at least five clonesshould be examined to have more than a 90% probability of haplotypingboth copies of the genomic locus in an individual. In some cases,however, once the haplotype for one genomic allele is directlydetermined, the haplotype for the other allele may be inferred if theindividual has a known genotype for the PSs of interest or if thehaplotype frequency or haplotype pair frequency for the individual'spopulation group is known.

While direct haplotyping of both copies of the gene is preferablyperformed with each copy of the gene being placed in separatecontainers, it is also envisioned that direct haplotyping could beperformed in the same container if the two copies are labeled withdifferent tags, or are otherwise separately distinguishable oridentifiable. For example, if first and second copies of the gene arelabeled with different first and second fluorescent dyes, respectively,and an allele-specific oligonucleotide labeled with yet a thirddifferent fluorescent dye is used to assay the PS(s), then detecting acombination of the first and third dyes would identify the polymorphismin the first gene copy while detecting a combination of the second andthird dyes would identify the polymorphism in the second gene copy.

The nucleic acid sample used in the above indirect and directhaplotyping methods is typically isolated from a biological sample takenfrom the individual, such as a blood sample or tissue sample. Suitabletissue samples include whole blood, saliva, tears, urine, skin and hair.

The target region(s) containing the PS of interest may be amplifiedusing any oligonucleotide-directed amplification method, including butnot limited to polymerase chain reaction (PCR) (U.S. Pat. No.4,965,188), ligase chain reaction (LCR) (Barany et al., Proc. Natl.Acad. Sci. USA 88:189-93 (1991); WO 90/01069), and oligonucleotideligation assay (OLA) (Landegren et al., Science 241:1077-80 (1988)).Other known nucleic acid amplification procedures may be used to amplifythe target region(s) including transcription-based amplification systems(U.S. Pat. No. 5,130,238; European Patent No. EP 329,822; U.S. Pat. No.5,169,766; WO 89/06700) and isothermal methods (Walker et al., Proc.Natl. Acad. Sci. USA 89:392-6 (1992)).

In both the direct and indirect haplotyping methods, the identity of anucleotide (or nucleotide pair) at a PS(s) in the amplified targetregion may be determined by sequencing the amplified region(s) usingconventional methods. If both copies of the gene are represented in theamplified target, it will be readily appreciated by the skilled artisanthat only one nucleotide will be detected at a PS in individuals who arehomozygous at that site, while two different nucleotides will bedetected if the individual is heterozygous for that site. Thepolymorphism may be identified directly, known as positive-typeidentification, or by inference, referred to as negative-typeidentification. For example, where a polymorphism is known to be guanineand cytosine in a reference population, a site may be positivelydetermined to be either guanine or cytosine for an individual homozygousat that site, or both guanine and cytosine, if the individual isheterozygous at that site. Alternatively, the site may be negativelydetermined to be not guanine (and thus cytosine/cytosine) or notcytosine (and thus guanine/guanine).

A PS in the target region may also be assayed before or afteramplification using one of several hybridization-based methods known inthe art. Typically, allele-specific oligonucleotides are utilized inperforming such methods. The allele-specific oligonucleotides may beused as differently labeled probe pairs, with one member of the pairshowing a perfect match to one variant of a target sequence and theother member showing a perfect match to a different variant. In someembodiments, more than one PS may be detected at once using a set ofallele-specific oligonucleotides or oligonucleotide pairs. Preferably,the members of the set have melting temperatures within 5° C., and morepreferably within 2° C., of each other when hybridizing to each of thepolymorphic sites being detected.

Hybridization of an allele-specific oligonucleotide to a targetpolynucleotide may be performed with both entities in solution, or suchhybridization may be performed when either the oligonucleotide or thetarget polynucleotide is covalently or noncovalently affixed to a solidsupport. Attachment may be mediated, for example, by antibody-antigeninteractions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,hydrophobic interactions, chemical linkages, UV cross-linking baking,etc. Allele-specific oligonucleotides may be synthesized directly on thesolid support or attached to the solid support subsequent to synthesis.Solid-supports suitable for use in detection methods of the inventioninclude substrates made of silicon, glass, plastic, paper and the like,which may be formed, for example, into wells (as in 96-well plates),slides, sheets, membranes, fibers, chips, dishes, and beads. The solidsupport may be treated, coated or derivatized to facilitate theimmobilization of the allele-specific oligonucleotide or target nucleicacid.

Detecting the nucleotide or nucleotide pair at a PS of interest may alsobe determined using a mismatch detection technique, including but notlimited to the RNase protection method using riboprobes (Winter et al.,Proc. Natl. Acad. Sci. USA 82:7575 (1985); Meyers et al., Science230:1242 (1985)) and proteins which recognize nucleotide mismatches,such as the E. coli mutS protein (Modrich, Ann. Rev. Genet. 25:229-53(1991)). Alternatively, variant alleles can be identified by singlestrand conformation polymorphism (SSCP) analysis (Orita et al., Genomics5:874-9 (1989); Humphries et al., in MOLECULAR DIAGNOSIS OF GENETICDISEASES, Elles, ed., pp. 321-340, 1996) or denaturing gradient gelelectrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-706(1990); Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-6 (1989)).

A polymerase-mediated primer extension method may also be used toidentify the polymorphism(s). Several such methods have been describedin the patent and scientific literature and include the “Genetic BitAnalysis” method (WO 92/15712) and the ligase/polymerase mediatedgenetic bit analysis (U.S. Pat. No. 5,679,524. Related methods aredisclosed in WO 91/02087, WO 90/09455, WO 95/17676, and U.S. Pat. Nos.5,302,509 and 5,945,283. Extended primers containing the complement ofthe polymorphism may be detected by mass spectrometry as described inU.S. Pat. No. 5,605,798. Another primer extension method isallele-specific PCR (Ruaño et al., 1989, supra; Ruaño et al., 1991,supra; WO 93/22456; Turki et al., J. Clin. Invest. 95:1635-41 (1995)).In addition, multiple PSs may be investigated by simultaneouslyamplifying multiple regions of the nucleic acid using sets ofallele-specific primers as described in WO 89/10414.

The genotype or haplotype for the CDK5 gene of an individual may also bedetermined by hybridization of a nucleic acid sample containing one orboth copies of the gene, mRNA, cDNA or fragment(s) thereof, to nucleicacid arrays and subarrays such as described in WO 95/11995. The arrayswould contain a battery of allele-specific oligonucleotides representingeach of the PSs to be included in the genotype or haplotype.

The invention also provides a kit for determining whether an individualhas a response marker I, a response marker II, or a response marker III.The kit comprises a set of one or more oligonucleotides designed foridentifying at least one of the alleles at each PS in a set of one ormore PSs, wherein the set of one or more PSs comprises (a) PS3 and PS4;(b) PS2, PS3, and PS4; (c) a set of one or more PSs in a linkedhaplotype for any of haplotypes (1)-(2) in Table 1, or (d) a set of oneor more PSs in a substitute haplotype for any of haplotypes (1)-(2) inTable 1. Preferably, the kit comprises a set of one or moreoligonucleotides designed for identifying at least one of the alleles ateach PS in a set of one or more PSs, wherein the set of one or more PSsis any of (a) PS3 and PS4; (b) PS2, PS3, and PS4; (c) a set of one ormore PSs in a linked haplotype for any of haplotypes (1)-(2) in Table 1,and (d) a set of one or more PSs in a substitute haplotype for any ofhaplotypes (1)-(2) in Table 1.

In a preferred embodiment of the kit of the invention, the set of one ormore oligonucleotides is designed for identifying both alleles at eachPS in the set of one or more PSs. In another preferred embodiment, theindividual is Caucasian. In another preferred embodiment, the kitfurther comprises a manual with instructions for (a) performing one ormore reactions on a human nucleic acid sample to identify the allele oralleles present in the individual at each PS in the set of one or morePSs, and (b) determining if the individual has a response marker I, aresponse marker II, or a response marker III based on the identifiedallele or alleles. In another preferred embodiment, the linkagedisequilibrium between a linked haplotype for any of haplotypes (1)-(2)in Table 1 and any of haplotypes (1)-(2) in Table 1 has a delta squaredvalue selected from the group consisting of at least 0.75, at least0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0. In yetanother preferred embodiment, the linkage disequilibrium between anallele at a substituting PS and an allele at a substituted PS for any ofhaplotypes (1)-(2) in Table 1 has a delta squared value selected fromthe group consisting of at least 0.75, at least 0.80, at least 0.85, atleast 0.90, at least 0.95, and 1.0.

As used herein, an “oligonucleotide” is a probe or primer capable ofhybridizing to a target region that contains, or that is located closeto, a PS of interest. Preferably, the oligonucleotide has less thanabout 100 nucleotides. More preferably, the oligonucleotide is 10 to 35nucleotides long. Even more preferably, the oligonucleotide is between15 and 30, and most preferably, between 20 and 25 nucleotides in length.The exact length of the oligonucleotide will depend on the nature of thegenomic region containing the PS as well as the genotyping assay to beperformed and is readily determined by the skilled artisan.

The oligonucleotides used to practice the invention may be comprised ofany phosphorylation state of ribonucleotides, deoxyribonucleotides, andacyclic nucleotide derivatives, and other functionally equivalentderivatives. Alternatively, oligonucleotides may have a phosphate-freebackbone, which may be comprised of linkages such as carboxymethyl,acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and thelike (Varma, in MOLECULAR BIOLOGY AND BIOTECHNOLOGY, A COMPREHENSIVEDESK REFERENCE, Meyers, ed., pp. 617-20, VCH Publishers, Inc., 1995).Oligonucleotides of the invention may be prepared by chemical synthesisusing any suitable methodology known in the art, or may be derived froma biological sample, for example, by restriction digestion. Theoligonucleotides may be labeled, according to any technique known in theart, including use of radiolabels, fluorescent labels, enzymatic labels,proteins, haptens, antibodies, sequence tags and the like.

Oligonucleotides of the invention must be capable of specificallyhybridizing to a target region of a polynucleotide containing a desiredlocus. As used herein, specific hybridization means the oligonucleotideforms an anti-parallel double-stranded structure with the target regionunder certain hybridizing conditions, while failing to form such astructure when incubated with another region in the polynucleotide orwith a polynucleotide lacking the desired locus under the samehybridizing conditions. Preferably, the oligonucleotide specificallyhybridizes to the target region under conventional high stringencyconditions.

A nucleic acid molecule such as an oligonucleotide or polynucleotide issaid to be a “perfect” or “complete” complement of another nucleic acidmolecule if every nucleotide of one of the molecules is complementary tothe nucleotide at the corresponding position of the other molecule. Anucleic acid molecule is “substantially complementary” to anothermolecule if it hybridizes to that molecule with sufficient stability toremain in a duplex form under conventional low-stringency conditions.Conventional hybridization conditions are described, for example, inMOLECULAR CLONING, A LABORATORY MANUAL, 2^(nd) ed., Sambrook et al.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and in NUCLEICACID HYBRIDIZATION, A PRACTICAL APPROACH, Haymes et al., IRL Press,Washington, D.C., 1985. While perfectly complementary oligonucleotidesare preferred for detecting polymorphisms, departures from completecomplementarity are contemplated where such departures do not preventthe molecule from specifically hybridizing to the target region. Forexample, an oligonucleotide primer may have a non-complementary fragmentat its 5′ end, with the remainder of the primer being complementary tothe target region. Alternatively, non-complementary nucleotides may beinterspersed into the probe or primer as long as the resulting probe orprimer is still capable of specifically hybridizing to the targetregion.

Preferred oligonucleotides of the invention, useful in determining if anindividual has a response marker I, a response marker II, or a responsemarker III, are allele-specific oligonucleotides. As used herein, theterm allele-specific oligonucleotide (ASO) means an oligonucleotide thatis able, under sufficiently stringent conditions, to hybridizespecifically to one allele of a gene, or other locus, at a target regioncontaining a PS while not hybridizing to the corresponding region inanother allele(s). As understood by the skilled artisan,allele-specificity will depend upon a variety of readily optimizedstringency conditions, including salt and formamide concentrations, aswell as temperatures for both the hybridization and washing steps.Examples of hybridization and washing conditions typically used for ASOprobes are found in Kogan et al., “Genetic Prediction of Hemophilia A”in PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, Academic Press,1990, and Ruaño et al., Proc. Natl. Acad. Sci. USA 87:6296-300 (1990).Typically, an ASO will be perfectly complementary to one allele whilecontaining a single mismatch for another allele.

Allele-specific oligonucleotides of the invention include ASO probes andASO primers. ASO probes which usually provide good discriminationbetween different alleles are those in which a central position of theoligonucleotide probe aligns with the polymorphic site in the targetregion (e.g., approximately the 7^(th) or 8^(th) position in a 15mer,the 8^(th) or 9^(th) position in a 16mer, and the 10^(th) or 11^(th)position in a 20mer). An ASO primer of the invention has a 3′ terminalnucleotide, or preferably a 3′ penultimate nucleotide, that iscomplementary to only one of the nucleotide alleles of a particular SNP,thereby acting as a primer for polymerase-mediated extension only ifthat nucleotide allele is present at the PS in the sample beinggenotyped. ASO probes and primers hybridizing to either the coding ornoncoding strand are contemplated by the invention. ASO probes andprimers listed below use the appropriate nucleotide symbol (R=G or A,Y=T or C, M=A or C, K=G or T, S=G or C, and W=A or T; WIPO standardST.25) at the position of the PS to represent that the ASO containseither of the two alternative allelic variants observed at that PS.

A preferred ASO probe for detecting the alleles at each of PS2, PS3, andPS4, is listed in Table 4. Additionally, detection of the alleles ateach of PS2, PS3, and PS4 could be accomplished by utilization of thecomplement of these ASO probes.

A preferred ASO forward and reverse primer for detecting the alleles ateach of PS2, PS3, and PS4 is listed in Table 4.

TABLE 4 Preferred ASOs for Detecting Alleles at PSs in HaplotypesComprising Preferred Embodiments of Response Markers I and ResponseMarkers II¹ ASO ASO ASO Probe Forward Primer Reverse Primer SEQ SEQ SEQNucleotide ID Nucleotide ID Nucleotide ID PS sequence NO. sequence NO.sequence NO. 2 GGGCACAST 2 GTTGCTGGGCA 5 CCTGAACATG 8 TGCATG CAST CAAST3 AGAGTCCRT 3 GAGGGGAGAGT 6 GCTCAAAACT 9 GGAGTT CCRT CCAYG 4 TGCAGCTKT 4TGAAGATGCAG 7 TGGGAAAGGG 10  GGCCCT CTKT CCAMA ¹These ASO probes andprimers include the appropriate nucleotide symbol, Y = T or C, R = G orA, M = A or C, K = G or T/U, and S = G or C (World Intellectual PropertyOrganization Handbook on Industrial Property Information andDocumentation IPO Standard ST.25 (1998), Appendix 2, Table 1), at theposition of the PS to represent that the ASO contains one of the twoalternative polymorphisms observed at that position.

Other oligonucleotides useful in practicing the invention hybridize to atarget region located one to several nucleotides downstream of a PS in aresponse marker. Such oligonucleotides are useful in polymerase-mediatedprimer-extension methods for detecting an allele at one of the PSs inthe markers described herein and therefore such oligonucleotides arereferred to herein as “primer-extension oligonucleotides.” In apreferred embodiment, the 3′-terminus of a primer-extensionoligonucleotide is a deoxynucleotide complementary to the nucleotidelocated immediately adjacent to the PS. A particularly preferred forwardand reverse primer-extension oligonucleotide for detecting the allelesat each of PS2, PS3, and PS4 is listed in Table 5. Termination mixes arechosen to terminate extension of the oligonucleotide at the PS ofinterest, or one base thereafter, depending on the alternativenucleotides present at the PS.

TABLE 5 Preferred Primer Extension Oligos for Detecting Alleles at PSsin Haplotypes Comprising Preferred Embodiments of Response Markers I andResponse Markers II Forward Reverse Primer Extension Primer Extension PSSequence SEQ ID NO. Sequence SEQ ID NO. 2 GCTGGGCACA 11 GAACATGCAA 14 3GGGAGAGTCC 12 CAAAACTCCA 15 4 AGATGCAGCT 13 GAAAGGGCCA 16

In some embodiments, the oligonucleotides in a kit of the invention havedifferent labels to allow probing of the identity of nucleotides ornucleotide pairs at two or more PSs simultaneously.

The oligonucleotides in a kit of the invention may also be immobilizedon or synthesized on a solid surface such as a microchip, bead, or glassslide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilizedoligonucleotides may be used in a variety of polymorphism detectionassays, including but not limited to probe hybridization and polymeraseextension assays. Immobilized oligonucleotides useful in practicing theinvention may comprise an ordered array of oligonucleotides designed torapidly screen a nucleic acid sample for polymorphisms in multiple genesat the same time.

Kits of the invention may also contain other components such ashybridization buffer (e.g., where the oligonucleotides are to be used asallele-specific probes) or dideoxynucleotide triphosphates (ddNTPs;e.g., where the alleles at the polymorphic sites are to be detected byprimer extension). In a preferred embodiment, the set ofoligonucleotides consists of primer-extension oligonucleotides. The kitmay also contain a polymerase and a reaction buffer optimized forprimer-extension mediated by the polymerase. Preferred kits may alsoinclude detection reagents, such as biotin- or fluorescent-taggedoligonucleotides or ddNTPs and/or an enzyme-labeled antibody and one ormore substrates that generate a detectable signal when acted on by theenzyme. It will be understood by the skilled artisan that the set ofoligonucleotides and reagents for performing the genotyping orhaplotyping assay will be provided in separate receptacles placed in thecontainer if appropriate to preserve biological or chemical activity andenable proper use in the assay.

In a particularly preferred embodiment, each of the oligonucleotides andall other reagents in the kit have been quality tested for optimalperformance in an assay for determining the alleles at a set of PSscomprising a response marker I, a response marker II, or a responsemarker III.

In addition to the kits, the invention provides a method for predictingthe cognitive response of an individual to treatment with a galantamine.The method comprises determining whether the individual has a responsemarker I, a response marker II, or a response marker III, and making aresponse prediction based on the results of the determining step. Thedetermination of the response marker present in an individual can bemade using one of the direct or indirect methods described herein. Insome preferred embodiments, the determining step comprises identifyingfor one or both copies of the genomic locus present in the individualthe identity of the nucleotide or nucleotide pair at the set of PSscomprising the selected response marker. Alternatively, the determiningstep may comprise consulting a data repository that states theindividual's copy number for the haplotypes comprising one of theresponse markers I, response markers II, or response markers III. Thedata repository may be the individual's medical records or a medicaldata card. In preferred embodiments, the individual is Caucasian.

In some embodiments, if the individual is determined to have a responsemarker I, then the response prediction is that the individual is morelikely to respond to galantamine than an individual having a responsemarker II or a response marker III, if the individual is determined tohave a response marker II, then the response prediction is that theindividual is less likely to respond to galantamine than an individualhaving a response marker I, but more likely to respond to galantaminethan an individual having a response marker III, and if the individualis determined to have a response marker III, then the responseprediction is that the individual is less likely to response togalantamine than an individual having a response marker I or a responsemarker II.

In other aspects, the invention provides an article of manufacture. Inone embodiment, an article of manufacture comprises a pharmaceuticalformulation and at least one indicium identifying a population for whichthe pharmaceutical formulation is indicated, wherein the identifiedpopulation has a cognitive disorder. The pharmaceutical formulationcomprises a galantamine compound as at least one active ingredient.Additionally, the pharmaceutical formulation may be regulated and theindicium may comprise the approved label for the pharmaceuticalformulation. The identified population is partially or wholly defined byhaving a response marker I or a response marker II, wherein a trialpopulation having a response marker I is more likely to respond to theformulation than a trial population lacking a response marker I, and atrial population having a response marker II is more likely to respondto the formulation than a trial population lacking both a responsemarker I and a response marker II. The identified population preferablymay be further defined as Caucasian. A population wholly defined byhaving a response marker I or a response marker II is one for whichthere are no other factors which should be considered in identifying thepopulation for which the pharmaceutical formulation is indicated. Incontrast, a population that is partially defined by having a responsemarker I or a response marker II is one for which other factors may bepertinent to identification of the population for which thepharmaceutical formulation is indicated. Examples of other such factorsare age, weight, gender, disease state, possession of other geneticmarkers or biomarkers, or the like. The cognitive disorder can includemild or moderate dementia of the Alzheimer's type, and dementiaassociated with Parkinson's Disease.

The pharmaceutical formulation may be formulated, in any way known inthe art, for any mode of delivery (i.e., oral), and any mode of release(i.e., sustained release). In some embodiments, the pharmaceuticalformulation is a tablet or capsule and the article may further comprisean additional indicium comprising the color or shape of the table orcapsule. In other embodiments, the article may further comprise anadditional indicium comprising a symbol stamped on the tablet orcapsule, or a symbol or logo printed on the approved label.

In some embodiments of this article, the approved label may comprise astatement about the identified population. In some or all of theseembodiments, the label may describe the change in cognitive functionexpected for the identified population. Additionally, in some or all ofthese embodiments, a galantamine is present in the pharmaceuticalformulation at an amount effective to improve cognitive function in theidentified population. The galantamine compound that is present in thepharmaceutical formulation is selected from galantamine, a galantaminederivative, and pharmaceutically acceptable salts of galantamine or thegalantamine derivative. Various galantamine derivatives have beenreported to be useful for treating Alzheimer's and related dementias,including but not limited to the compounds described and claimed in U.S.Pat. Nos. 6,150,354, 6,268,358, 6,319,919 B1, 6,323,196, and 6,326,196;and the compounds described and claimed in European Patent ApplicationNo. EP 236684. Pharmaceutically acceptable salts of galantamine reportedto be useful in treating Alzheimer's disease and related dementiasinclude those described in U.S. Pat. Nos. 4,663,318 and 6,358,941, aswell as WO 00/38686. In preferred embodiments, the galantamine compoundis galantamine hydrobromide.

An additional embodiment of the article of manufacture provided by theinvention comprises packaging material and a pharmaceutical formulationcontained within said packaging material. The pharmaceutical formulationcomprises a galantamine compound as at least one active ingredient. Thepackaging material may comprise a label stating that the pharmaceuticalformulation is indicated for a population having a cognitive disorder,wherein the population is partly or wholly defined by having a responsemarker I or a response marker II. The indicated population preferablymay be further defined as Caucasian. The label may further state that aspecified test can be used to identify members of the indicatedpopulation. Preferably the specified test is a genetic test. Thecognitive disorder can include mild or moderate dementia of theAlzheimer's type, and dementia associated with Parkinson's Disease.

Additionally, in other aspects of the invention, a method ofmanufacturing a drug product comprising a galantamine compound as atleast one active ingredient is provided. The method comprises combiningin a package a pharmaceutical formulation comprising the galantaminecompound and a label that states that the formulation is indicated for apopulation having a cognitive disorder, wherein the population ispartially or wholly defined by having a response marker I or a responsemarker II, wherein a trial population having a response marker I is morelikely to respond to the formulation than a trial population lacking aresponse marker I, and a trial population having a response marker II ismore likely to respond to the formulation than a trial populationlacking both a response marker I and a response marker II. The indicatedpopulation may be identified on the pharmaceutical formulation, on thelabel or on the package by at least one indicium, such as a symbol orlogo, color, or the like. The indicated population preferably may befurther defined as Caucasian. The cognitive disorder can include mild ormoderate dementia of the Alzheimer's type, and dementia associated withParkinson's Disease. The galantamine compound is selected fromgalantamine, a galantamine derivative, and pharmaceutically acceptablesalts of galantamine or the galantamine derivative.

Detecting the presence of a response marker I, a response marker II, ora response marker III in an individual is also useful in a method forseeking regulatory approval for marketing a pharmaceutical formulationfor improving cognitive function in a population having a cognitivedisorder, wherein the population is partially or wholly defined byhaving a response marker I or a response marker II. The method comprisesconducting at least one clinical trial which comprises administering thepharmaceutical formulation and a placebo to each of a first, second,third, and fourth treatment group of individuals having a cognitivedisorder, wherein each individual in the first treatment group has aresponse marker I, each individual in the second treatment group lacks aresponse marker I, each individual in the third treatment group has aresponse marker II, and each individual in the fourth treatment grouphas a response marker III, demonstrating that the first treatment groupis more likely to respond to the pharmaceutical formulation than thesecond treatment group, demonstrating that the third treatment group ismore likely to respond to the pharmaceutical formulation than the fourthtreatment group, and filing with a regulatory agency an application formarketing approval of the pharmaceutical formulation with a labelstating that the pharmaceutical formulation is indicated for apopulation having a cognitive disorder, and further stating thatindividuals having a response marker I are more likely to respond to thepharmaceutical formulation than individuals having a response marker IIor a response marker III, and that individuals having a response markerII are more likely to respond to the pharmaceutical formulation thanindividuals having a response marker III. In preferred embodiments, theregulatory agency is the United States Food and Drug Administration(FDA) or the European Agency for the Evaluation of Medicinal Products(EMEA), or a future equivalent of these agencies.

The clinical trial may be conducted by recruiting individuals having acognitive disorder, determining whether or not they have a responsemarker I, a response marker II, or a response marker III, and assigningthem to the first, second, third, and fourth treatment groups based onthe results of the determining step. The individuals in each treatmentgroup are preferably administered the same dose of the pharmaceuticalformulation, which includes, as at least one active ingredient, acompound effective in improving cognitive function, such as agalantamine compound, including galantamine, a galantamine derivative,and pharmaceutically acceptable salts of galantamine or the galantaminederivative. The pharmaceutical formulation may contain other activeingredients, for example another compound known or believed to beeffective in improving cognitive function. The cognitive disorder caninclude mild or moderate dementia of the Alzheimer's type, and dementiaassociated with Parkinson's Disease.

The regulatory agency may be any person or group authorized by thegovernment of a country anywhere in the world to control the marketingor distribution of drugs in that country. Preferably, the regulatoryagency is authorized by the government of a major industrializedcountry, such as Australia, Canada, China, a member of the EuropeanUnion, Japan, and the like. Most preferably the regulatory agency isauthorized by the government of the United States and the type ofapplication for approval that is filed will depend on the legalrequirements set forth in the last enacted version of the Food, Drug andCosmetic Act that are applicable for the pharmaceutical formulation andmay also include other considerations such as the cost of making theregulatory filing and the marketing strategy for the composition. Forexample, if the pharmaceutical formulation has previously been approvedfor the same cognitive function, then the application might be a paperNDA, a supplemental NDA or an abbreviated NDA, but the application wouldbe a full NDA if the pharmaceutical formulation has never been approvedbefore; with these terms having the meanings applied to them by thoseskilled in the pharmaceutical arts or as defined in the Drug PriceCompetition and Patent Term Restoration Act of 1984.

Additionally, in other aspects of the invention, there is provided amethod for marketing a drug product comprising promoting to a targetaudience the use of a drug product for improving cognitive function in apopulation having a cognitive disorder, wherein the population ispartially or wholly defined by having a response marker I or a responsemarker II, wherein the drug product comprises a compound effective inimproving cognitive function, and wherein a trial population having aresponse marker I are more likely to respond to the drug product than atrial population lacking a response marker I, and a trial populationhaving a response marker II are more likely to respond to the drugproduct than a trial population lacking both a response marker I and aresponse marker III. The drug product can comprise any compoundeffective in improving cognitive function, such as a galantaminecompound, including galantamine, a galantamine derivative, andpharmaceutically acceptable salts of galantamine or the galantaminederivative. The target audience can be members of a group that is inposition to influence prescription or purchase of the drug product. Suchgroups include physicians, pharmacists, insurance companies and healthmaintenance organizations, individuals at risk for developing AD, andgovernment agencies such as those involved in providing or regulatingmedical insurance and those involved in regulating the marketing ofdrugs.

The promoting step can employ printed publications such as medicaljournals and consumer magazines, radio and television advertisements,and public presentations such as presentations at medical and scientificconferences. In a preferred embodiment, the drug product is approved formarketing to delay the onset of AD in the population, and the promotingstep includes a statement that relates the approved drug product to itsappearance, e.g., the color or shape of a tablet or capsule formulation,or some design stamped or embossed thereon.

In practicing any of the embodiments of the invention that are describedherein, determination of the therapeutically effective dose of agalantamine compound and/or the appropriate route of administration iswell within the capability of those skilled in the art. For example, thedose can be estimated initially either in cell culture assays or in ananimal model of the cognitive disorder. Such information may then beused to determine the approximate concentration range and route ofadministration for humans. The exact dosage will be determined by thepractitioner, in light of factors relating to the patient requiringtreatment, including but not limited to severity of the disease state,general health, age, weight and gender of the patient, diet, time andfrequency of administration, other drugs being taken by the patient, andtolerance/response to the treatment.

One known animal model for Alzheimer's disease in humans is described inHaroutunian et al., Life Sciences 37:945-52 (1985). This rat model has aselective lesion placed in a subcortical nucleus (nucleus basalis ofMeynert), which results in a cortical cholinergic deficiency, similar inmagnitude to that seen in early to moderate stage Alzheimer's disease.Numerous behavioral deficits, including the inability to learn andretain new information, characterizes this lesion. Drugs that cannormalize these abnormalities would have a reasonable expectation ofefficacy in Alzheimer's disease.

The galantamine compound or composition used in practicing the inventionmay be administered to a patient orally or by subcutaneous orintravenous injection. Sustained release delivery mechanisms may beparticularly useful, for example, intracerebroventricularly by means ofan implanted reservoir by use of sustained release capsules or by meansof a transdermal patch. It may be necessary to begin at lower doses thanare ultimately effective.

Certain galantamine compounds used in practicing different embodimentsof the invention may be only sparingly soluble in water at roomtemperature and so injectable compositions are normally in the form ofan aqueous suspension. If necessary, pharmaceutically-acceptablesuspension aids may be employed. Typically, such a suspension will beemployed at a concentration of 1-50 mg/ml more commonly 5-40 mg/ml, forexample, 5-30 mg/ml or 10-40 mg/ml, typically 20-30 mg/ml of thegalantamine compound of interest.

Typical dosage rates when administering a galantamine compound willdepend upon the activity of the compound and the exact nature andcondition of the patient. For example, typical dosage rates foradministration by injection are in the range 5-1,000 mg per daydepending upon the patient. In some cases, even lower dosages such as0.5 or 1 mg per day may be helpful. For example, divided doses in therange 0.5-5 mg/kg body weight per day may prove useful. Typically, onemight administer a dosage of 50-300 mg per day to a patient of a bodyweight of 40-100 kg, although in appropriate cases such dosages mayprove useful for patients having a body weight outside this range. Inother cases, dosages as low as 0.1 mg and as high as 500 mg may beappropriate for persons in this body weight range.

Galantamine compounds used in practicing the invention may also beadministered orally, for example, as an aqueous suspension or a solutionin aqueous ethanol or as a solid such as a tablet or capsule.Suspensions or solutions for oral administration are typically of aboutthe same concentration as those used for injections. However, it may bedesirable when administering the drug orally to use a higher dosage ratethan when administering it by injection. For example, dosages up to 200mg per day may be used, such as dosages in the range 10-60 mg per day.In preparing such tablets or capsules, standard tablet or capsule-makingtechniques may be employed. The dosage rate of the compound of theinvention or its pharmaceutically-acceptable salt will normally be inthe same range as for oral administration of a liquid. If desired, apharmaceutically-acceptable carrier such as starch or lactose may beused in preparing tablets. Capsules may be prepared using soft gelatinas the encapsulating agent. If desired, such capsules may be in the formof sustained release capsules wherein the main capsule containsmicrocapsules of active compound which release the contents over aperiod of several hours thereby maintaining a constant level of activecompound in the patient's blood stream.

The following specific formulations may find use in practicing one ormore embodiments of the present invention: (1) Tablets or capsulescontaining 0.1, 0.5, 1.0, 5, 10 and 25 mg of the hydrobromide salt ofgalantamine or a galantamine derivative to be taken four times a day, ora sustained-release-preparation delivering an equivalent daily dose; (2)a parenteral solution containing 5 mg/ml of the galantamine compound;and (3) a liquid formulation for oral administration available in 5 mg/5ml and 25 mg/5 ml concentration.

There have been reports that galantamine can cause cardiac arrhythmias.If such problems are believed to be a risk when practicing an embodimentof the present invention, it may be desirable to administer thegalantamine compound in conjunction with another drug such aspropantheline bromide to control such arrhythmias. Since other sideeffects, such as nausea, are common with drugs that act on the centralnervous system, a galantamine compound or composition used in thepresent invention may be administered in conjunction with an agent forcontrol of such side effects.

Further, in performing any of the methods described herein which requireinformation on the haplotype content of the individual (i.e., thehaplotypes and haplotype copy number present in the individual for thepolymorphic sites in haplotypes comprising a response marker I, aresponse marker II, or a response marker III) or which require knowingif a response marker I, a response marker II, or a response marker IIIis present in the individual, the individual's CDK5 haplotype content orresponse marker may be determined by consulting a data repository suchas the individual's patient records, a medical data card, a file (e.g.,a flat ASCII file) accessible by a computer or other electronic ornon-electronic media on which information about the individual's CDK5haplotype content or response marker can be stored. As used herein, amedical data card is a portable storage device such as a magnetic datacard, a smart card, which has an on-board processing unit and which issold by vendors such as Siemens of Munich Germany, or a flash-memorycard. The medical data card may be, but does not have to be, credit-cardsized so that it easily fits into pocketbooks, wallets and other suchobjects carried by the individual. The medical data card may be swipedthrough a device designed to access information stored on the data card.In an alternative embodiment, portable data storage devices other thandata cards can be used. For example, a touch-memory device, such as the“i-button” produced by Dallas Semiconductor of Dallas, Tex. can storeinformation about an individual's CDK5 haplotype content or responsemarker, and this device can be incorporated into objects such asjewelry. The data storage device may be implemented so that it canwirelessly communicate with routing/intelligence devices through IEEE802.11 wireless networking technology or through other methods wellknown to the skilled artisan. Further, as stated above, informationabout an individual's haplotype content or response marker can also bestored in a file accessible by a computer; such files may be located onvarious media, including: a server, a client, a hard disk, a CD, a DVD,a personal digital assistant such as a Palm Pilot, a tape, a zip disk,the computer's internal ROM (read-only-memory) or the internet orworldwide web. Other media for the storage of files accessible by acomputer will be obvious to one skilled in the art.

Any or all analytical and mathematical operations involved in practicingthe methods of the present invention may be implemented by a computer.For example, the computer may execute a program that assigns CDK5haplotype pairs and/or a response marker I, a response marker II, or aresponse marker III to individuals based on genotype data inputted by alaboratory technician or treating physician. In addition, the computermay output the predicted change in cognitive function in response to agalantamine following input of the individual's CDK5 haplotype contentor response marker, which was either determined by the computer programor input by the technician or physician. Data on which response markerswere detected in an individual may be stored as part of a relationaldatabase (e.g., an instance of an Oracle database or a set of ASCII flatfiles) containing other clinical and/or haplotype data for theindividual. These data may be stored on the computer's hard drive ormay, for example, be stored on a CD ROM or on one or more other storagedevices accessible by the computer. For example, the data may be storedon one or more databases in communication with the computer via anetwork.

It is also contemplated that the above described methods andcompositions of the invention may be utilized in combination withidentifying genotype(s) and/or haplotype(s) for other genomic regions.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims that follow the examples.

EXAMPLES

The Examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the scope ofthe invention in any way. The Examples do not include detaileddescriptions for conventional methods employed, such as in the synthesisof oligonucleotides or polymerase chain reaction. Such methods are wellknown to those skilled in the art and are described in numerouspublications, for example, MOLECULAR CLONING: A LABORATORY MANUAL,2^(nd) ed., supra.

Example 1

This example illustrates the clinical and biochemical characterizationof selected individuals in a cohort of 449 Caucasian patients diagnosedwith Alzheimer's Disease.

The patient cohort was selected from patients participating in threeclinical trials of galantamine held internationally and in the UnitedStates (GAL-INT2, GAL-USA 10, and GAL-INT-1) (Rockwood et al., supra;Tariot et al., supra; Wilcock et al., supra), and a fourth clinicaltrial with a similar disease population. In brief, the galantaminetrials were carried out by delivering to patients galantamine at dailydosages of 8 mg, 16 mg, 24 mg, or 32 mg depending on the trial.Following 3, 5, 6 or 12 months of treatment in the GAL-INT2, GAL-USA 10,GAL-INT-1 and SAB-USA-25 trials, respectively, the severity of symptomsin patients were evaluated using the cognitive subscale of theAlzheimer's Disease Assessment Scale (ADAS-cog) (Rosen et al., supra;Rockwood et al., supra; Tariot et al., supra; Wilcock et al., supra).The ADAS-cog measures cognitive function, including spoken languageability, comprehension of spoken language, recall of test instructions,word-finding difficulty in spontaneous speech, following commands,naming objects and fingers, constructional praxis, ideational praxis,orientation, word-recall task and word-recognition task (Alzheimer'sInsights Online, supra).

For the clinical association study described in Example 2 below, 174patients were selected and used to populate two groups in a tailedsampling strategy, intended to enrich alleles correlating with drugresponse in the population. This population consisted of 85 respondersand 89 non-responders. Patients were assigned to responder andnon-responder groups based on having a change in ADAS-cog score(ΔADAS-cog) that met a cut-off value that was chosen based on thedifferences in treatment times in the four clinical trials describedabove. The ΔADAS-cog cut-off values and treatment times used for eachresponder and non-responder group from each of the four clinical trialsare shown in Table 6 below.

TABLE 6 ΔADAS-cog Used to Select Patients for Responder andNon-Responder Groups Treatment Time Clinical Trial (months) ResponderNon-responder GAL-INT-2 3 Δ ≦ −5 Δ ≧ 2 GAL-USA-10 5 Δ ≦ −7 Δ ≧ 3GAL-INT-1 6 Δ ≦ −7 Δ ≧ 3 SAB-USA-25 12 Δ ≦ −3 N/ATable 7 below shows the number of patients from each of the fourclinical trials that were placed in each of the clinical associationanalyses groups.

TABLE 7 Composition of the Treatment Group Trial Name (Number ofTreatment Group Patients) Responder Non-Responder Total GALINT1 0 0 0GALINT2 4 1 5 GALUSA10 81 88 169 SABUSA25 0 0 0 TOTAL 85 89 174

Example 2

This example illustrates genotyping of the patient cohort for the fourCDK5 polymorphic sites selected by the inventors herein for analysis.

Genomic DNA samples were isolated from blood samples obtained from eachmember of the cohort and genotyped at each of PS1-PS4 (Table 2) usingthe MassARRAY technology licensed from Sequenom (San Diego, Calif.). Inbrief, this genotyping technology involves performing a homogeneousMassEXTEND assay (hME), in which an initial polymerase chain reaction isfollowed by an allele-specific oligonucleotide extension reaction in thesame tube or plate well, and then detecting the extended oligonucleotideby MALDI-TOF mass spectrometry.

For each of the four CDK5 polymorphic sites of interest, a genomic DNAsample was amplified in a 8.0 μL multiplexed PCR reaction consisting of2.5 ng genomic DNA (0.3 ng/μL), 0.85 μL 10× reaction buffer, 0.32 unitsTaq Polymerase, up to five sets of 0.4 pmol each of forward PCR primer(5′ to 3′) and reverse PCR primer (3′ to 5′) and 1.6 nmol each of dATP,dCTP, dGTP and dTTP. A total of four reactions were performed comprisingthe following polymorphic site groups: (1) PS1; (2) PS2; (3) PS3; and(4) PS4. Forward and Reverse PCR primers used for each of the four CDK5polymorphic sites consisted of a 10 base universal tag(5′-AGCGGATAAC-3′; SEQ ID NO:17) followed by one of the CDK5-specificsequences shown in Tables 8A and 8B below:

TABLE 8A Forward PCR CDK5-specific Primer Sequences used in hME AssaysPS1 AGCGGATAACTTCTACCGCGGAGGCAAAC (SEQ ID NO:18) PS2AGCGGATAACAATGACTGGGAGGAGAGAGG (SEQ ID NO:19) PS3AGCGGATAACATCACTGCCCTCACCCATTG (SEQ ID NO:20) PS4AGCGGATAACCTCACCCTGCCCCTGAAGAT (SEQ ID NO:21)

TABLE 8B Reverse PCR CDK5-specific Primer Sequences used in hME AssaysPS1 AGCGGATAACAAACGGACACGCGTTGCTTC (SEQ ID NO:22) PS2AGCGGATAACAACCAGGTGCTGAAAGGTGG (SEQ ID NO:23) PS3AGCGGATAACTATCCTTGCTTTCCTCTGCC (SEQ ID NO:24) PS4AGCGGATAACGGAGTCAAGGATCACTTGGG (SEQ ID NO:25)

PCR thermocycling conditions were: initial denaturation of 95° C. for 15minutes followed by 45 cycles of 94° C. for 20 seconds, 56° C. for 30seconds and 72° C. for 1 minute followed by a final extension of 72° C.for 3 minutes. Following the final extension, unincorporateddeoxynucleotides were degraded by adding 0.48 units of Shrimp AlkalinePhosphatase (SAP) to the PCR reactions and incubation for 20 minutes at37° C. followed by 5 minutes at 85° C. to inactivate the SAP.

Template-dependent primer extension reactions were then performed on themultiplexed PCR products by adding a 2.0 μL volume of an hME cocktailconsisting of 720 pmol each of three dideoxynucleotides and 720 pmol ofone deoxynucleotide, 8.6 pmol of an extension primer, 0.2 μL of 5×Thermosequenase Reaction Buffer, and NanoPure grade water. Thethermocycling conditions for the mass extension reaction were: initialdenaturation for 2 minutes at 94° C. followed by 40 cycles of 94° C. for5 seconds, 40° C. for 5 seconds and 72° C. for 5 seconds. Extensionprimers used to genotype each of the four CDK5 polymorphic sites areshown in Table 9 below:

TABLE 9 Extension Primers for Genotyping CDK5 Polymorphic Sites PS1GGAGGCAAACCTTGGACTTCAA (SEQ ID NO:26) PS2 TCAGACGCCCTGAACATGCAA (SEQ IDNO:27) PS3 TCACCCATTGTGCTCAAAACTCCA (SEQ ID NO:28) PS4 CCCCTGAAGATGCAGCT(SEQ ID NO:29)

The extension products were desalted prior to analysis by massspectrometry by mixing them with AG50X8 NH₄OAc cation exchange resin.

The desalted multiplexed extension products were applied onto aSpectroCHIP™ using the SpectroPOINT™ 24 pin applicator tool as permanufacturer's instructions (Sequenom Industrial Genomics, Inc. SanDiego, Calif.). The SpectroChip™ was loaded into a Bruker Biflex III™linear time-of flight mass spectrometer equipped with a SCOUT 384 ionsource and data was acquired using XACQ 4.0, MOCTL 2.1, AutoXecute 4.2and XMASS/XTOF 5.0.1 software on an Ultra 5™ work station (SunMicrosystems, Palo Alto Calif.). Mass spectrometry data was subsequentlyanalyzed on a PC running Windows NT 4.0 (Microsoft, Seattle Wash.) withSpectroTYPER™ genotype calling software (Sequenom Industrial Genomics,Inc. San Diego, Calif.).

Example 3

This example illustrates the deduction of haplotypes from the CDK5genotyping data generated in Example 2.

Haplotypes were estimated from the unphased genotypes using acomputer-implemented algorithm for assigning haplotypes to unrelatedindividuals in a population sample, essentially as described in WO01/80156 (Genaissance Pharmaceuticals, Inc., New Haven, Conn.). In thismethod, haplotypes are assigned directly from individuals who arehomozygous at all sites or heterozygous at no more than one of thevariable sites. This list of haplotypes is then used to deconvolute theunphased genotypes in the remaining (multiply heterozygous) individuals.

A quality control analysis was performed on the deduced haplotypes,which included analysis of the frequencies of the haplotypes andindividual SNPs therein for compliance with principles of Hardy-Weinbergequilibrium.

Example 4

This example illustrates analysis of the CDK5 haplotypes in Table 1 forassociation with individuals' responses to galantamine.

The statistical analyses compared ΔADAS-cog in patients with one copyvs. zero copies, two copies vs. zero copies, and two copies vs. one copy(within a patient's genome) of a particular allele, using a logisticregression analysis on two-degrees of freedom to associate clinicalresponse with a particular haplotype. The following covariates were alsoincluded: age, gender, history, smoking, ADAS-cog baseline, dose (BID),body mass index, and CYP2D6. The logistic regression included assessmentof associations between the haplotypes and the binary outcome ofclinical response.

For the results obtained on the analyses, adjustments were made formultiple comparisons, using a permutation test (MULTIVARIATE PERMUTATIONTESTS: WITH APPLICATIONS IN BIOSTATISTICS, Pesarin, John Wiley and Sons,New York, 2001). In this test, a sub-haplotype's data for eachobservation were kept constant, while all the remaining variables(outcome and covariates) were randomly permuted so that covariatesalways stayed with the same outcome. The permutation model was fittedfor each of the several haplotypes, and the lowest p-value was kept. Intotal, 1000 permutations were done. Two CDK5 haplotypes of at least onepolymorphism were identified that show a correlation with anindividual's ability to respond to galantamine. These CDK5 haplotypesare shown above in Table 1, and the unadjusted (“raw”) and adjusted(“perm.”) p-values for these two haplotypes are shown below in Tables10A, 10B, and 10C.

TABLE 10A CDK5 Haplotypes Having Association with Response toGalantamine (1 vs. 0 copies) Subject Count Lower for HaplotypeConfidence Subject Count with Highest Odds Interval Perm. Raw forHaplotype Level Response Ratio (C.I.) of Upper C.I. Haplotype p p (# ofcopies) (# of copies) (O.R.) O.R. of O.R. (1) 0.012 0.002477 88 (1) 46(1) 0.5067927 0.2248439 1.1422984 41 (0) 26 (0) (2) 0.012 0.002477 88(1) 46 (1) 0.5067927 0.2248439 1.1422984 41 (0) 26 (0)

TABLE 10B CDK5 Haplotypes Having Association with Response toGalantamine (2 vs. 0 copies) Subject Count Lower for HaplotypeConfidence Subject Count with Highest Odds Interval Perm. Raw forHaplotype Level Response Ratio (C.I.) of Upper C.I. Haplotype p p (# ofcopies) (# of copies) (O.R.) O.R. of O.R. (1) 0.012 0.002477 45 (2) 13(2) 0.183118 0.0695317 0.4822576 41 (0) 26 (0) (2) 0.012 0.002477 45 (2)13 (2) 0.183118 0.0695317 0.4822576 41 (0) 26 (0)

TABLE IOC CDK5 Haplotypes Having Association with Response toGalantamine (2 vs. 1 copies) Subject Count Lower for HaplotypeConfidence Subject Count with Highest Odds Interval Perm. Raw forHaplotype Level Response Ratio (C.I.) of Upper C.I. Haplotype p p (# ofcopies) (# of copies) (O.R.) O.R. of O.R. (1) 0.012 0.002477 45 (2) 13(2) 0.3613272 0.1581388 1.8255869 88 (1) 46 (1) (2) 0.012 0.002477 45(2) 13 (2) 0.3613272 0.1581388 1.8255869 88 (1) 46 (1)

As seen in Tables 10A, 10B, and 10C, each of haplotypes (1) and (2)shows a correlation with an individual's response to galantamine. Theodds ratio (O.R.) column for Table 10A indicates the likelihood that anindividual with one copy of haplotype (1) or haplotype (2) will respondto galantamine as compared to an individual with zero copies ofhaplotype (1) or haplotype (2), wherein an O.R. greater than 1 indicatesthat an individual with one copy is more likely to respond than anindividual with zero copies, and an O.R. less than 1 indicates that anindividual with one copy is less likely to respond than an individualwith zero copies. The O.R. column for Table 10B indicates the likelihoodthat an individual with two copies of haplotype (1) or haplotype (2)will respond to galantamine as compared to an individual with zerocopies of haplotype (1) or haplotype (2), wherein an O.R. greater than 1indicates that an individual with two copies is more likely to respondthan an individual with zero copies, and an O.R. less than 1 indicatesthat an individual with two copies is less likely to respond than anindividual with zero copies. The O.R. column for Table 10C indicates thelikelihood that an individual with two copies of haplotype (1) orhaplotype (2) will respond to galantamine as compared to an individualwith one copy of haplotype (1) or haplotype (2), wherein an O.R. greaterthan 1 indicates that an individual with two copies is more likely torespond than an individual with one copy, and an O.R. less than 1indicates that an individual with two copies is less likely to respondthan an individual with one copy.

In summary, the study described herein identified CDK5 haplotypes thatare correlated with the likelihood of whether an individual will exhibita cognitive response to galantamine. It is believed that suchinformation will be useful to physicians in deciding whether a patientshould be prescribed galantamine for treating AD and other diseases thatcause dementia or cognitive impairment, in performing clinical trials ofgalantamine and derivatives thereof, and in obtaining marketing approvalof galantamine for treating diseases that cause cognitive impairment.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained. Asvarious changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification, including patents and patentapplications, are hereby incorporated in their entirety by reference.The discussion of references herein is intended merely to summarize theassertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

1.-28. (canceled)
 29. A kit for determining whether an individual has aresponse marker I, a response marker II, or a response marker III, thekit comprising a set of one or more oligonucleotides designed foridentifying at least one of the alleles at each polymorphic site (PS) ina set of one or more PSs, wherein the set of one or more PSs comprises:(a) PS3 and PS4; (b) PS2, PS3, and PS4; (c) a set of one or more PSs ina linked haplotype for any of haplotypes (1)-(2) in Table 1; or (d) aset of one or more PSs in a substitute haplotype for any of haplotypes(1)-(2) in Table 1, wherein the enumerated PSs in sets (a)-(b)correspond to the following nucleotide positions in SEQ ID NO:1: PS2,3892; PS3, 4808; and PS4,
 5284. 30. The kit of claim 29, wherein the kitcomprises a set of one or more oligonucleotides designed for identifyingat least one of the alleles at each PS in a set of one or more PSs,wherein the set of one or more PSs is any of: (a) PS3 and PS4; (b) PS2,PS3, and PS4; (c) a set of one or more PSs in a linked haplotype for anyof haplotypes (1)-(2) in Table 1; and (d) a set of one or more PSs in asubstitute haplotype for any of haplotypes (1)-(2) in Table 1, whereinthe enumerated PSs in sets (a)-(b) correspond to the followingnucleotide positions in SEQ ID NO:1: PS2, 3892; PS3, 4808; and PS4,5284.
 31. The kit of claim 29, wherein the set of one or moreoligonucleotides is designed for identifying both alleles at each PS inthe set of one or more PSs.
 32. The kit of claim 29, wherein the set ofone or more PSs is (a), (c), or (d), wherein if the set is (c), then thelinked haplotype is a linked haplotype for haplotype (1) in Table 1, andwherein if the set is (d), then the substitute haplotype is a substitutehaplotype for haplotype (1) in Table
 1. 33. The kit of claim 32, whereinthe set of one or more PSs is (a).
 34. The kit of claim 29, wherein theindividual is Caucasian.
 35. The kit of claim 29, which furthercomprises a manual with instructions for (a) performing one or morereactions on a human nucleic acid sample to identify the allele oralleles present in the individual at each PS in the set of one or morePSs, and (b) determining if the individual has a response marker I, aresponse marker II, or a response marker III based on the identifiedallele or alleles.
 36. The kit of claim 29, wherein the linkagedisequilibrium between the linked haplotype and at least one ofhaplotypes (1)-(2) in Table 1 has a delta squared value selected fromthe group consisting of at least 0.75, at least 0.80, at least 0.85, atleast 0.90, at least 0.95, and 1.0.
 37. The kit of claim 29, wherein theset of one or more PSs is (a) or (c), wherein if the set is (c), thenthe linked haplotype is a linked haplotype for haplotype (1) in Table 1and the linkage disequilibrium between the linked haplotype andhaplotype (1) in Table 1 has a delta squared value of at least 0.95. 38.The kit of claim 29, wherein the linkage disequilibrium between theallele at a substituting PS in the substitute haplotype and the alleleat a substituted PS in any of haplotypes (1)-(2) in Table 1 has a deltasquared value selected from the group consisting of at least 0.75, atleast 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0. 39.The kit of claim 29, wherein the set of one or more PSs is (a) or (d),wherein if the set is (d), then the substitute haplotype is a substitutehaplotype for haplotype (1) in Table 1 and the linkage disequilibriumbetween the allele at a substituting PS in the substitute haplotype andthe allele at a substituted PS in haplotype (1) in Table 1 has a deltasquared value of at least 0.95.
 40. The kit of claim 29, wherein atleast one oligonucleotide in the set of one or more oligonucleotides isan allele-specific oligonucleotide (ASO) probe comprising a nucleotidesequence, wherein the sequence is any of SEQ ID NOS:2-4 and theircomplements.
 41. The kit of claim 40, wherein the set of one or more PSsis (a) and the at least one oligonucleotide in the set of one or moreoligonucleotides is a first ASO probe, a second ASO probe, a third ASOprobe, and a fourth ASO probe, wherein the first ASO probe comprises anucleotide sequence, wherein the sequence is SEQ ID NO:3 or itscomplement, wherein R in SEQ ID NO:3 is G, and wherein the second ASOprobe comprises a nucleotide sequence, wherein the sequence is SEQ IDNO:3 or its complement, wherein R in SEQ ID NO:3 is A, wherein the thirdASO probe comprises a nucleotide sequence, wherein the sequence is SEQID NO:4 or its complement, wherein K in SEQ ID NO:4 is G, wherein thefourth ASO probe comprises a nucleotide sequence, wherein the sequenceis SEQ ID NO:4 or its complement, wherein K in SEQ ID NO:4 is T.
 42. Thekit of claim 29, wherein at least one oligonucleotide in the set of oneor more oligonucleotides is a primer-extension oligonucleotidecomprising a nucleotide sequence, wherein the sequence is any of SEQ IDNOS:5-16.
 43. The kit of claim 42, wherein the set of one or more PSs is(a) and the at least one oligonucleotide in the set of one or moreoligonucleotides is a first primer-extension oligonucleotide and asecond primer-extension oligonucleotide, wherein the first primerextension oligonucleotide comprises a nucleotide sequence, wherein thesequence is any of SEQ ID NO:12 and SEQ ID NO:15, and wherein the secondprimer-extension oligonucleotide comprises a nucleotide sequence,wherein the sequence is any of SEQ ID NO:13 and SEQ ID NO:16. 44.-76.(canceled)